ABSTRACT Antibiotic resistance is a worldwide problem, which threatens to disarm important procedures in modern medicine, such as cancer therapy, organ transplantation, etc. P. aeruginosa is a significant source of hospital acquired infections and the leading cause of mortality in patients with cystic fibrosis. To combat antibiotic resistance new antibiotics and new targets are needed. We propose to validate iron homeostasis as a new target for future development of antibiotics. Bacterial iron homeostasis offers a significant vulnerability because essential iron must be obtained from the host, which makes the nutrient scarce to invading pathogens (nutritional immunity). Pathogens have evolved mechanisms to ?steal? iron from their host, but these depend on well-regulated iron homeostasis. We are targeting the protein/protein interactions between the iron storage protein bacterioferritin (BfrB) and its associated ferredoxin (Bfd), which are necessary to regulate cytosolic iron concentrations. Importantly, BfrB and Bfd exist only in bacteria. The proposed work builds from our crystal structure of the BfrB:Bfd complex, and from having shown that blocking the BfrB:Bfd interaction disrupts iron homeostasis, causes P. aeruginosa cells to become iron deficient, and significantly less virulent in C. elegans model of infection. Our approach is multidisciplinary and involves investigators with expertise in bacterial iron metabolism and structural biology (Rivera), organic synthesis/drug discovery (Bunce), chemical biology and medicinal chemistry (Peterson), and microbiology (Chandler). The aims are to: 1) Develop small molecule probes for blocking the BfrB:Bfd interaction in vitro and in P. aeruginosa. (A) We will utilize structure based-design principles to develop promising molecules obtained from screening a small library into potent inhibitors of the BfrB:Bfd interaction. (B) Screen a large library to find additional molecules that bind BfrB. These will be subjected to co-crystallization trials with BfrB, and the emerging structural information used to guide their synthetic elaboration into probes capable of blocking the BfrB:Bfd interaction. 2) Study the consequences of perturbing the BfrB:Bfd interaction in P. aeruginosa using genetic and chemical intervention. We will use genetic techniques to interrogate the effects of blocking the BfrB:Bfd interaction on iron homeostasis in P. aeruginosa. As we learn about these effects using gene deletions and mutations to the chromosome, we will use the emerging information as a benchmark to evaluate the efficacy of the chemical probes developed in Aim 1. The new probes will serve as the first-ever molecular tools for interrogating bacterial iron homeostasis, first in a model organism such as P. aeruginosa PAO1, then in clinical isolates of P. aeruginosa, and ultimately in other pathogenic bacteria where the BfrB:Bfd interaction is conserved.